Method for transferring one or more genetic traits from a plant of the purple-flowered capsicum species to a plant of the white-flowered capsicum species

ABSTRACT

The present invention relates to a method for transferring one or more genetic traits from a purple-flowered  Capsicum  species plant comprising said genetic trait, to a white-flowered  Capsicum  species plant, said method comprising the steps of: (a) preparing a first hybrid plant comprising said genetic trait by crossing a first purple-flowered  Capsicum  species comprising said genetic traits with a second purple-flowered  Capsicum  species; (b) preparing a second hybrid plant by crossing said first hybrid plant with a first white-flowered  Capsicum  species plant; (c) crossing said second hybrid plant with said first purple-flowered  Capsicum  species plant to produce first progeny plants; and (d) preparing a third hybrid plant by crossing the first progeny plants obtained in step (c) with a second white-flowered  Capsicum  species plant to produce second progeny plants, and selecting the third hybrid plant from the second progeny plants comprising said one or more genetic traits.

CROSS-REFERENCE

This application is a divisional of U.S. Utility Application Ser. No.11/992,503, now U.S. Pat. No. 8,022,278, which was filed on May 19,2008, which is a national stage application filed under 35 U.S.C. 371 ofInternational Application No. PCT/EP2005/054759 filed on Sep. 22, 2005,each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for transferring one or moregenetic traits from a plant of the purple-flowered Capsicum speciescomprising-said genetic trait, to a plant of the white-flowered Capsicumspecies. The invention further relates to the plants per se, as well asto the fruits, seeds and other plant parts derived from said plants.

BACKGROUND OF THE INVENTION

The genus Capsicum belongs to the large family of the Solanaceae.Several species of the Capsicum group are valuable crops that are grownin open fields or under protected conditions in many countries all overthe world. Capsicum species with pungent fruits are generally used as aspice, either fresh, dry or extracted. Species with nonpungent fruits ina big variety of shapes and colours, commonly known as sweet peppers,are widely used as vegetable.

The genus Capsicum can be divided in two distinct groups based on theflower colour: the white-flowered group, comprising e.g. the closelyrelated species C. annuum, C. frutescens, C. baccatum, C. chacoense, C.galapagoense, C. practermissum and C. chinense, and the purple-floweredgroup, comprising e.g. C. tovarii, C. pubescens, C. eximium and C.cardenasii. Of the white-flowered group Capsicum annuum, and its closelyrelated species C. frutescens are the best known domesticated species,which are mainly used for the production of sweet and hot peppers forprocessing and consumption.

Capsicum, like other plants, is attacked by many pests and diseases,including insects, nematodes, fungi, bacteria and viruses. One of suchbacterial diseases is bacterial leafspot, caused by the bacteriumXanthomonas campestris pv. vesicatoria. Bacterial leafspot of Capsicumcan be recognized by numerous angular spot on the leaves. Initially, thespots are water-soaked. Leaves infected at an early stage becomedeformed. Often the margins of affected leaves are rimmed with a narrowband of necrotic tissue. Infected Capsicum leaves drop prematurely, bywhich the fruit is exposed to sun which may result in sun scald,secondary fruit rots and reduced yields. Capsicum fruits rarely showsymptoms but may drop if infected early.

X. vesicatoria is widespread and damaging to Capsicum in particular infield-grown crops in warm-temperate and tropical countries, and thus hasa large economic impact. Control of the disease relies principally onthe production of plants from healthy (treated) seeds and on preventivesteps taken during the entire season.

Useful resistances to such diseases, as well as other traits such asfruit quality characteristics etc., may exist in wild or otherdomesticated species of Capsicum, such as for example in C. pubescenswhich carries unique and specific traits, such as the resistance againstcertain races of the bacterium Xanthomonas vesicatoria, that are notpresent in the white flowering species. It thus may be advantageous totransfer such favourable genetic traits, such as any resistance gene,from the distinctly related purple-flowered C. pubescens to the commonlyused white-flowered species such as C. annuum and/or C. frutescens.

It is well known, however, that strong crossing barriers of varyingdegree exist between the different species of the Capsicum genus. Somecrosses have even been proven impossible, and other crosses are onlypossible with the help of in vitro culture techniques, such as embryorescue. In addition, in most cases the products derived from saidcrosses are completely or partially sterile which makes them notaccessible for any further breeding purposes.

A need therefore exists for a method by which desirable genetic traitscan be transferred between the different purple and white floweredCapsicum species.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method fortransferring one or more genetic traits, such as, but not limited to,resistance genes, from plants of the purple-flowered Capsicum species toplants of the white-flowered Capsicum species.

This is achieved by providing a method, comprising the steps of:

-   -   (a) preparing a first hybrid plant comprising said genetic trait        by crossing a first plant of the purple-flowered Capsicum        species comprising said genetic trait with a second plant of        another purple-flowered Capsicum species, and selecting from the        progeny thereof plants comprising said genetic trait;    -   (b) preparing a second hybrid plant by crossing said first        hybrid plant comprising said genetic trait, with a first plant        of the white-flowered Capsicum species;    -   (c) crossing said second hybrid plant with said first plant of        the purple-flowered Capsicum species comprising said genetic        trait, and selecting from the progeny thereof plants comprising        the genetic trait;    -   (d) preparing a third hybrid plant by crossing the plants        obtained step c) with a second plant of the white-flowered        Capsicum species, and selecting from the progeny thereof plants        comprising the genetic trait;

According to the present invention, for the first time a genetic traithas been successfully transferred from a purple-flowered Capsicumspecies to a white-flowered Capsicum species. The method of the presentinvention is based on a suitable combination of several interspecificcrosses, as well as crosses that serve as a kind of bridge to the nextspecies (FIG. 1). The desired genetic traits can for example be followedby molecular markers or bio-assay according to well-known molecularbiological techniques known to the skilled person.

According to a preferred embodiment of the present invention, the methodfurther comprises:

-   -   (e) selecting plants obtained in step (d) not comprising the        genetic trait and crossing these plants with said first plant of        the purple-flowered Capsicum species comprising said genetic        trait, and selecting from the progeny thereof plants comprising        the genetic trait; and    -   (f) preparing a fourth hybrid plant by crossing the plants        obtained step (e) with a second plant of the white-flowered        Capsicum species, and selecting from the progeny thereof plants        comprising the genetic trait. In this way, plants that are        obtained in step (d) of the method of the invention that do not        comprise the genetic trait, still can be used further in the        method of the invention.

According to a further preferred embodiment, the method furthercomprises:

-   -   (g) preparing a fifth hybrid plant by crossing the fourth hybrid        plant comprising the genetic trait with a second plant of the        white-flowered Capsicum species, and selecting from the progeny        thereof plants comprising the genetic trait. Thus, a stable        white-flowered plant is obtained comprising the genetic trait of        interest derived from the purple-flowered species but with all        the quality aspects of the white-flowered species.

Preferably, after step (b) the second hybrid plant is self-pollinatedand the progeny thereof is used in step (c) in order to fix theresistance, to improve fertility aspects and to obtain homozygous,genetically stable plants carrying the desired trait(s).

According to another preferred embodiment, the plants obtained in step(c) are self-pollinated and the progeny thereof is used in step (d),again in order to fix the resistance, to improve fertility aspects andto obtain homozygous, genetically stable plants carrying the desiredtrait(s).

In addition, according to other preferred embodiments, the plantsobtained in step (d) are self-pollinated and the progeny thereof is usedin step (e), and/or the plants obtained in step (e) are self-pollinatedand the progeny thereof is used in step (f).

According to the present invention embryo rescue techniques are appliedwhenever it is beneficial to the process in terms of obtaining hybridplants or speeding up the process.

Preferably, the first plant of the purple-flowered Capsicum species isselected from the group consisting of C. pubescens, C. eximium, and C.cardenasii, more preferably the first plant of the purple-floweredCapsicum species is C. pubescens. The greatest genetic distance in thegenus Capsicum is between the species C. pubescens and C.annuum/frutescens. Direct crosses between these species have been provenimpossible until now, even with the help of embryo rescue. By using themethod of the present invention, however, a totally new gene poolbecomes accessible to the species of C. annuum and/or C. frutescens.

According to the invention, the second plant of the purple-floweredCapsicum species is selected from the group consisting of C. pubescens,C. eximium, and C. cardenasii, preferably the second plant of thepurple-flowered Capsicum species is C. eximium or C. cardenasii,preferably C. eximium. Preferably, the first plant is selected fromanother species than the second plant of the purple-flowered Capsicumspecies.

In a preferred embodiment of the invention, the first plant of thewhite-flowered Capsicum species is selected from the group consisting ofC. baccatum, C. frutescens, C. chinense, C. annuum, and hybrids thereof.Preferably, the first plant of the white-flowered Capsicum species is C.frutescens.

The second plant of the white-flowered Capsicum species is preferablyselected from the group consisting of C. baccatum, C. frutescens, C.chinense and C. annuum, and hybrids thereof, more preferably the secondplant of the white-flowered Capsicum species is C. chinense, C. annuumand/or a hybrid thereof.

According to the present invention a hybrid is a plant obtained from across between two different species of the Capsicum genus, e.g. a crossbetween a plant of the species C. chinense and a plant of the species C.annuum (interspecific cross), or a plant obtained from a cross betweenpopulations or cultivars of a single species (intraspecific cross).

According to a further preferred embodiment of the invention, thegenetic trait is a resistance gene against a specific Capsicum diseaseattacking all cultivated species of Capsicum. By transferring one ormore resistance genes from distinctly related species of the Capsicumgenus to commonly used species not naturally having such resistancegenes, new resistant plants are provided. As a consequence, the use ofenvironmental undesirable chemical treatments may for example be reducedor even abandoned. However, also other genetic traits, such as otherresistance genes, fruit quality characteristics such as shape, taste andcolour, improved yield, fruit set, and resistance against abiotic stress(e.g. cold/salt) may be transferred using the method of the presentinvention.

In a particular preferred embodiment, the resistance gene is the Bs4gene, conveying resistance to the bacterial disease Xanthomanascampestris pv. vesicatoria race I, II, IV and VI.

The present invention further relates to plants, obtainable by themethod as described above.

In particular, the invention relates to plants of the white-floweredCapsicum species, comprising a genetic trait derived from a plant of thepurple-flowered Capsicum species said plants comprising a genetic traitwhich normally is not present in the white-flowered species.

In a preferred embodiment, the plant is selected from the groupconsisting of C. baccatum, C. frutescens, C. chinense, C. annuum, andhybrids thereof, preferably, the plant is C. chinense, C. annuum and/orhybrids thereof.

According to a preferred embodiment of the invention, the genetic traitis a resistance gene against a Capsicum disease, in particular theresistance gene is the Bs4 gene, conveying resistance to the bacterialdisease Xanthomanas campestris pv. vesicatoria race I, II, IV and VI.

Furthermore, the present invention relates to fruits, seeds and otherplant parts derived from the plants described above, wherein the otherplant parts may be selected from the group consisting of seeds,cuttings, runners, and meristem.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by the following Example andFigures, which are not intended to limit the scope of the invention inany way.

FIG. 1 schematically shows the steps of the method of the presentinvention;

FIGS. 2A-2B are photographs of a C. eximium plant (A) and a C. pubescensPI235047 (B) plant with the desired resistance gene used in a first stepof a preferred embodiment of the method of the invention;

FIG. 3A shows an example of a first hybrid plant, i.e. the productobtained in the first step of the method of the invention; FIG. 3B is aphotograph of C. frutescens_P1238059;

FIGS. 4A-4C show examples of a second hybrid plant (A), and photographsof C. annuum (B) and C. chinense (C);

FIG. 5 is a photograph of the product of the self-pollinated ((C.pubescens×C. eximium)×C. frutescens)×(C. chinense×C. annuum); and

FIGS. 6A-6D show C. annuum plants carrying the resistance genes derivedfrom C. pubescens.

DETAILED DESCRIPTION OF THE INVENTION Example

Methods:

Seeds were sown on vermiculate, potted into rockwool, blocks, andfertilized and propagated until flowering. Flowerbuds were emasculatedand pollinated with the chosen fatherlines. Between 24-28 days afterpollination fruits were harvested, surface sterilised and opened understerile conditions. Embryos from all seeds were isolated aseptically andtransferred to embryo rescue (ER) medium as described by Sibi et al.,(Aim. Amelior. Plantes, 29: 583-606, 1979). After 1-2 weeks welldeveloped plants were transferred to MS medium (Murashige T. and SkoogF., Physiol. Plant 15: 473497, 1962), supplemented with 0.8% agar and 20g/l sucrose. Plants with a well developed root system were subsequentlytransferred to the greenhouse and adapted carefully to its conditions.

Leaves of 4-6 weeks old plants were detached and inoculated withXanthomonas campestris vesicatoria race VI to select the resistantplants. The bacterial clone is stored in the freezer and grown on agarmedium one week before inoculation at 22° C. The suspension forinoculation is prepared by resuspending the bacteria in demineralisedwater. The bacteria suspension was applied to the leaves with a syringe.Inoculated leaves are kept in boxes covered with glass in a climate roomat 22° C. for two days. Leaves of resistant plants show a light brownand dry spot at the site of inoculation caused by a hypersensitivityreaction (HR). Leaves of susceptible plants show no reaction or a watersoaked lesion at the site of inoculation depending on the time ofincubation. Absence of a hypersensitivity reaction thus is indicative ofthe absence of the Bs4 gene. Resistant control is C. pubescens P1235047,susceptible control is a susceptible C. pubescens accession.

Crosses:

A first hybrid plant was prepared by crossing Capsicum pubescensP1235047 (male, FIG. 2B) (United states Department of Agriculture),comprising the Bs4 resistance gene, with Capsicum eximium (female, FIG.2A). 37 hybrid plants (FIG. 3A), comprising the resistance gene, wereobtained.

A second hybrid plant was subsequently prepared by crossing C.frutescens PI 238059 (male, FIG. 3B) with the first hybrid plantscomprising the resistance gene (female). None of the second hyrid plantsplants derived from this cross expressed the resistance.

Subsequently, the second hybrid plant (female, FIG. 4A) was crossed withCapsicum pubescens P1235047 (male). Two resistant plants were obtained.These plants were fertile and accordingly F2 seeds were obtained.

In addition, hybrids of C. annuum (FIG. 4B) and C. chinense (FIG. 4C)were made by crossing individuals of C. chinense PBC306 (Asian VegetableResearch and Development Center) with individuals of a C. annuum plant.The product of this cross was again crossed with C. annuum. SubsequentlyF2 seeds were obtained.

Subsequently, a third hybrid plant (FIG. 5) was prepared by crossing theresistant plants derived from the cross of the second hybrid plant withC. pubescens or plants derived from the F2 seeds thereof, with plantsderived from the F2 seeds of the C. annuum/C. chinense hybrid. Theresistant products from this cross were further crossed with pure C.annuum lines.

In addition, plants derived from said cross that were susceptible forXcv6, were crossed again with C. pubescens PI235047. The resistantplants derived from that cross were then selfed and F2 seeds wereobtained. Resistant plants derived from that cross were subsequentlyagain crossed with a line derived from the hybrid Abdera AMA12. Thishybrid is a cross between C. chinense and C. annuum. The resistantplants resulting from said last cross were selfed and F2 seeds wereobtained. In addition, the plants derived from the selfing of the lastmentioned cross with P1235047 were further crossed with several C.annuum parental lines.

As a result, C. annuum plants were obtained carrying the Bs4 resistancegene derived from C. pubescens (FIG. 6). These result clearly shows thatit is possible to introduce traits from the purple-flowered Capsicumspecies into the white-flowered Capsicum species, using the method ofthe present invention.

1-24. (canceled)
 25. A white-flowered Capsicum species plant, comprisinga genetic trait derived from a purple-flowered Capsicum species plant.26. The white-flowered Capsicum species plant as claimed in claim 25selected from the group consisting of: Capsicum baccatum, Capsicumfrutescens, Capsicum chinense, Capsicum annuum, and hybrids thereof. 27.The white-flowered Capsicum species plant as claimed in claim 26,wherein the plant is Capsicum chinense, Capsicum annuum or a hybridthereof.
 28. The white-flowered Capsicum species plant as claimed inclaim 25, wherein the one or more genetic traits is a resistance geneagainst a Capsicum disease.
 29. The plant as claimed in claim 28,wherein the resistance gene is the bacterial spot disease resistanceprotein 4 (Bs4) gene, conveying resistance to bacterial Xanthomanascampestris pv. vesicatoria race I, III and IV disease.
 30. Fruits, seedsor plant parts derived from the plant as claimed in claim 25.